CN111138540B

CN111138540B - Antibodies that bind FCRN for the treatment of autoimmune diseases

Info

Publication number: CN111138540B
Application number: CN202010031615.6A
Authority: CN
Inventors: 金圣旭; 朴胜国; 丁在甲; 安惠敬; 金旼善; 金银善; 龙海英; 申东玉; 宋连情; 柳泰亨
Original assignee: Hanall Biopharma Co Ltd
Current assignee: Hanall Biopharma Co Ltd
Priority date: 2014-04-30
Filing date: 2015-04-30
Publication date: 2023-07-25
Anticipated expiration: 2035-04-30
Also published as: DK3137504T3; JP2017522043A; WO2015167293A1; EP4241852A3; EP4241852A2; ES2952583T3; US20170210801A1; AU2015253915A1; NZ737666A; AU2018203582B2; KR101954906B1; CA3095295C; EA038470B1; MX2021005193A; AU2018203582A1; CN106459215A; SA516380194B1; KR20180093128A; MX2016014210A; EP3137504A4

Abstract

The present disclosure relates to antibodies that bind FCRN for the treatment of autoimmune diseases, in particular, to isolated anti-FCRN antibodies or fragments thereof, methods of making, compositions for the treatment of autoimmune diseases comprising and methods of using the antibodies to treat and diagnose autoimmune diseases, wherein the antibodies are antibodies that bind to FCRN (representing neonatal Fc receptor, also known as FcRP, fcRB or Brambell receptor), which is a receptor with high affinity for IgG. The FcRn-specific antibodies of the present disclosure bind FcRn non-competitively to IgG to reduce serum pathogenic autoantibody levels, and thus can be used to treat autoimmune diseases.

Description

Antibodies that bind FCRN for the treatment of autoimmune diseases

The present application is a divisional application of chinese patent application with application number 201580029793.2, application date 2015, month 4 and 30, entitled "antibodies binding FCRN for treatment of autoimmune diseases".

Technical Field

The present disclosure relates to isolated anti-FcRn antibodies, or fragments thereof, which are antibodies that bind to FcRn (representing a neonatal Fc receptor, also known as FcRP, fcRB or Brambell receptor), which is a receptor having high affinity for IgG, methods of making the same, compositions for treating autoimmune diseases comprising the antibodies, and methods of using the antibodies to treat and diagnose autoimmune diseases. The FcRn-specific antibodies of the present disclosure bind FcRn non-competitively to IgG to reduce serum pathogenic autoantibody levels, and thus can be used to treat autoimmune diseases.

Background

Antibodies are immune proteins that bind to a particular antigen. In most animals, including humans and mice, antibodies are composed of paired heavy and light chain polypeptides and each chain is composed of two distinct regions called a variable region and a constant region. The light chain variable region and the heavy chain variable region exhibit significant sequence diversity between antibodies and are responsible for binding to target antigens. The constant region shows less sequence diversity and is responsible for binding numerous native proteins to trigger important biochemical events.

Under normal conditions, the half-life of most human IgG (excluding IgG3 isotypes) in serum is about 22-23 days, which is longer relative to the serum half-life of other plasma proteins. For this longer serum half-life of IgG, igG entering the cell by endocytic processes can strongly bind to the neonatal Fc receptor (FcRn, an fcγ receptor) in the endosome at pH 6.0 to avoid the degradative lysosomal pathway. When the IgG-FcRn complex circulates to the plasma membrane, igG dissociates rapidly from FcRn in the blood stream at slightly alkaline pH (about 7.4). Through this receptor-mediated regeneration mechanism, fcRn effectively rescues IgG from degradation in lysosomes, thereby extending the half-life of IgG (Roopenian et al J. Immunol.170:3528,2003).

FcRn was identified in the digestive tract of neonatal rats, where it acts to mediate the absorption of IgG antibodies from maternal milk and promote their transport to the circulatory system. FcRn has also been isolated from the human placenta, where it mediates absorption of maternal IgG and transport to the fetal circulation. In adults, fcRn is expressed in numerous tissues, including lung, intestinal, renal epithelial tissues, and nasal, vaginal and biliary tract surfaces.

FcRn is a non-covalent heterodimer, typically found in endosomes of endothelial and epithelial cells. FcRn is a membrane-bound receptor with three heavy chain alpha domains (α1, α2, and α3) and a single soluble light chain β2-microglobulin (β2m) domain. Structurally, it belongs to the family of major histocompatibility complex class I molecules with β2m as the common light chain. The FcRn chain has a molecular weight of about 46kD and consists of an extracellular domain containing α1, α2 and α3 heavy chain domains and β2m light chain domain and having a single sugar chain, a single transmembrane and a relatively short cytoplasmic tail.

To study the contribution of FcRn to IgG homeostasis, mice have been engineered such that at least part of the genes encoding β2m and FcRn heavy chains have been "knocked out" so that these proteins are not expressed. In these mice, serum half-life and concentration of IgG were greatly reduced, suggesting an FcRn-dependent mechanism of IgG homeostasis. It has also been proposed that anti-human FcRn antibodies can be generated in these FcRn knockout mice and that these antibodies can prevent IgG binding to FcRn. Inhibition of IgG binding to FcRn adversely alters IgG serum half-life by preventing IgG regeneration, and thus autoimmune diseases caused by autoantibodies can be treated. This possibility is shown in a mouse model of autoimmune skin bullous disease (Li et al, J.Clin. Invest.115:3440,2005). Thus, agents that block or antagonize the binding of IgG to FcRn may be used in methods of treating or preventing IgG-mediated autoimmune and inflammatory diseases.

"autoimmune disease" encompasses a disease that occurs when the immune system of the body attacks its own normal tissues, organs or other in vivo components due to an immune system abnormality for which a cause cannot be found. These autoimmune diseases are systemic diseases that can occur in almost all parts of the body, including the nervous system, the gastrointestinal system, the endocrine system, the skin, the skeletal system and vascular tissue. Autoimmune diseases are known to affect about 5-8% of the world population, but reported prevalence of autoimmune diseases is lower than practical due to understanding autoimmune diseases and limitations in the methods of diagnosing these diseases.

The etiology of autoimmune diseases has been studied for a long time in terms of genetic, environmental and immune factors, but has not yet been clearly identified. Many recent studies have revealed that various autoimmune diseases are caused by IgG-type autoantibodies. Indeed, according to studies on the treatment of diseases and autoimmune diseases, the relationship between the presence or absence of disease-specific autoantibodies and the treatment of autoimmune diseases has been widely identified. Thus, the presence of disease-specific autoantibodies in a large number of autoimmune diseases and their pathological roles have been identified, and when the autoantibody of interest is removed from the blood, the effect of rapidly treating the disease can be obtained.

Autoimmune diseases and alloimmune diseases are mediated by pathogenic antibodies, and common examples thereof include immune neutropenia, guillain-Barre syndrome, epilepsy, autoimmune encephalitis, isaac syndrome, nevi syndrome, pemphigus vulgaris, deciduous pemphigus, bullous pemphigoid, acquired epidermolysis bullosa, gestational pemphigoid, mucosal pemphigoid, antiphospholipid syndrome, autoimmune anemia, autoimmune Grave's disease, goodpasture's syndrome, myasthenia gravis, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic Thrombocytopenic Purpura (ITP), lupus nephritis, or membranous nephropathy, or others.

For example, in the case of Myasthenia Gravis (MG), acetylcholine receptors (AChR) located at the neuromuscular junction of voluntary muscles are known to be destroyed or blocked by autoantibodies directed against said receptors, thereby disrupting voluntary muscle function. It is also known that when such autoantibodies are reduced, muscle function is restored.

In the case of ITP, ITP is a disease caused by destruction of peripheral platelets by the production of autoantibodies that bind to specific platelet membrane glycoproteins. Anti-platelet antibodies modulate platelets and cause rapid destruction of platelets by reticulocytes (e.g., macrophages).

In general, attempts to treat ITP include suppressing the immune system and thus causing elevated platelet levels. ITP affects women more frequently than men and is more common in children than adults. The incidence was 1/10,000 people. Chronic ITP is one of the major hematological diseases in adults and children. Chronic ITP generates significant hospitalization and treatment costs in the united states and the specialized hematologies worldwide. Approximately 20,000 new cases occur annually in the united states, and the costs of ITP care and special effects therapies are extremely high. Most ITP children have very low platelet counts resulting in sudden bleeding with common symptoms including bruising, reddish spots on the skin, epistaxis and gingival bleeding. While children may sometimes recover without treatment, many doctors recommend careful observation and relief of bleeding and gamma globulin intravenous infusion therapy.

An important pathogenesis of lupus nephritis, an autoimmune disease, is known to be the accumulation of increased immune complexes in systemic organs, which may occur due to inappropriate overproduction of autoantibodies, such as antinuclear antibodies, to cause inflammatory responses. About 40-70% of lupus patients develop kidney involvement, and about 30% of patients develop lupus nephritis, which is known to be an adverse prognostic factor for lupus patients. Although no treatment of lupus nephritis using immunosuppressants has been attempted, no remission is reported to be caused in about 22% of lupus nephritis patients, even when immunosuppressants are used. In addition, it is reported that 10-65% of patients relapse lupus nephritis when immunosuppressant use is reduced, even if remission is caused. Finally, 5-10% of patients with severe lupus nephritis (WHO grade III and IV) die after 10 years, and 5-15% enter the end-stage renal stage. Therefore, no suitable lupus nephritis therapy has been reported.

Thus, the use of antibodies with novel mechanisms for the treatment of autoimmune diseases by clearing pathogenic autoantibodies is expected to have therapeutic effects against pathogenic IgG-mediated autoimmune diseases (such as pemphigus vulgaris, neuromyelitis optica and myasthenia gravis), and immune complex-mediated glomerular diseases (such as lupus nephritis or membranous nephropathy).

Methods of treating autoimmune diseases by intravenous bulk administration of IgG (IVIG) have been widely used (Arnson Autoimmunity 42:42:553, 2009). The effects of IVIG are explained by a variety of mechanisms, and in addition by mechanisms that compete for FcRn with endogenous IgG to increase pathogenic antibody clearance. Intravenous administration of human immunoglobulin (IVIG) in large amounts has been shown to increase platelet count in infants suffering from immune ITP, and IVIG has been shown to be beneficial as a therapy for several other autoimmune disorders. Many studies have investigated the mechanism by which IVIG is effective in the treatment of autoimmune diseases. For ITP, early studies led to the following conclusions: IVIG effects are mainly due to the blocking effect on Fc receptors of platelets responsible for phagocytic antibody opsonization. Subsequent studies showed that Fc-depleted IVIG formulations caused elevated platelet counts in some ITP patients, and that IVIG effects were recently reported to be due to stimulation of fcyriib expression on macrophage cells, resulting in inhibition of the platelet phagocytosis process.

However, such IVIG treatments have a number of side effects and are very expensive to the user. In addition, other therapies for treating autoimmune/alloimmune disorders besides IVIG include polyclonal anti-D immunoglobulins, corticosteroids, immunosuppressants (including chemotherapeutics), cytokines, plasma separation, in vitro antibody adsorption (e.g., using a Prosorba column), surgical interventions such as splenectomy, and others. However, like IVIG, these therapies are complex, incomplete and costly. In addition, extremely high doses of IVIG are required to cause significant increases in pathogenic antibody clearance due to the putative mechanism by which IVIG inhibits pathogenic antibody binding to FcRn (i.e., competitive inhibition) and the fact that: igG shows very low affinity for FcRn at physiological pH (i.e., pH 7.2-7.4), and a common clinical dose of IVIG is about 2g/kg.

Treatment of autoimmune diseases with inhibitors that competitively inhibit IgG binding to FcRn is a promising treatment. However, due to the high affinity of endogenous IgG for FcRn and the high concentration of endogenous IgG in the blood, competitive inhibition of FcRn will likely require extremely high doses and thus have the same limitations as existing IVIG treatments.

Thus, although anti-FcRn antibodies are disclosed in WO 2006/118772, WO 2007/087289, WO 2009/131702, WO 2012/167039, there is an urgent need to develop improved human antibodies that have high affinity for FcRn and thus can remove pathogenic antibodies and reduce immunogenicity even at low doses.

Disclosure of Invention

Technical problem

The inventors have made intensive efforts to solve the above problems and to provide drugs effective and fundamentally treating autoimmune diseases (including ITP), and finally to provide antibodies or fragments thereof having high affinity for FcRn, and a method for preparing the same. Antibodies or fragments thereof that bind to FcRn specifically bind to the FcRn chain in a pH-independent manner and interfere in a non-competitive manner with the binding of the Fc of the antibody to FcRn, thereby treating autoimmune diseases by reducing in vivo autoantibodies that may cause the autoimmune disease.

It is an object of the present disclosure to provide a pharmaceutical composition for treating an autoimmune disease comprising an antibody that binds to FcRn, wherein the autoimmune disease is immune neutropenia (immune neutropenia), guillain-Barre syndrome (Guillain-Barre syndrome), epilepsy (epiepsy), autoimmune encephalitis (autoimmune encephalitis), isaac syndrome (Isaac's syndrome), nevus syndrome (nevus syndrome), pemphigus vulgaris (pemphigus vulgaris), deciduous pemphigus (Pemphigus foliaceus), bullous pemphigoid (Bullous pemphigoid), acquired bullous epidermolysis (epidermolysis bullosa acquisita), gestational pemphigoid (pemphigoid gestationis), mucosal pemphigoid (mucous membrane pemphigoid), antiphospholipid syndrome (antiphospholipid syndrome), autoimmune anemia (autoimmune anemia), autoimmune Graves's disease, goodpasture's syndrome (Goodpasture's syndrome), myasthenia gravis (6732), multiple sclerosis (multiple sclerosis), multiple sclerosis (3787), lupus erythematosus (3787), and other inflammatory diseases.

Technical solution

To achieve the above objects, the present disclosure provides an isolated anti-FcRn antibody or fragment thereof, the antibody comprising:

CDR1 comprising one or more amino acid sequences selected from SEQ ID nos. 21, 24, 27, 30, 33, 36, 39 and 42;

CDR2 comprising one or more amino acid sequences selected from SEQ ID nos. 22, 25, 28, 31, 34, 37, 40 and 43; and

CDR3 comprising one or more amino acid sequences selected from SEQ ID nos. 23, 26, 29, 32, 35, 38, 41 and 44.

In addition, the present disclosure provides an isolated anti-FcRn antibody or fragment thereof, the antibody comprising:

CDR1 comprising an amino acid sequence having at least 90% homology with one or more amino acid sequences selected from the group consisting of SEQ ID nos. 21, 24, 27, 30, 33, 36, 39 and 42;

CDR2 comprising an amino acid sequence having at least 90% homology with one or more amino acid sequences selected from the group consisting of SEQ ID nos. 22, 25, 28, 31, 34, 37, 40 and 43; and

CDR3 comprising an amino acid sequence having at least 90% homology with one or more amino acid sequences selected from the group consisting of SEQ ID nos. 23, 26, 29, 32, 35, 38, 41 and 44.

In addition, the present disclosure provides an isolated anti-FcRn antibody comprising one or more heavy chain variable regions and a light chain variable region comprising one or more amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID nos. 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.

In addition, the present disclosure provides an isolated anti-FcRn antibody comprising one or more heavy chain variable regions and a light chain variable region, the variable regions comprising an amino acid sequence having at least 90% homology to one or more amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID nos. 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.

In addition, the present disclosure provides polynucleotides encoding anti-FcRn antibodies or fragments thereof.

In addition, the present disclosure provides polynucleotides encoding anti-FcRn antibodies comprising one or more sequences selected from the group consisting of SEQ ID nos. 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19.

In addition, the present disclosure provides polynucleotides encoding anti-FcRn antibodies, the polynucleotides comprising sequences having at least 90% homology to one or more sequences selected from the group consisting of SEQ ID nos. 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19.

In addition, the present disclosure provides recombinant expression vectors comprising the polynucleotides, host cells transfected with the recombinant expression vectors. The present disclosure additionally provides a method of making an antibody or fragment thereof that specifically binds to FcRn, the method comprising: culturing the host cell and producing antibodies therefrom; and isolating and purifying the produced antibodies to recover the anti-FcRn antibodies.

In addition, the present disclosure provides a pharmaceutical composition comprising an anti-FcRn antibody or fragment thereof and one or more pharmaceutically acceptable carriers.

In addition, the present disclosure provides a method of treating a patient suffering from an autoimmune disease, the method comprising administering the composition to the patient.

In addition, the present disclosure provides a composition comprising an antibody labeled with a detection label.

In addition, the present disclosure provides a method of detecting FcRn in vivo or in vitro, the method comprising using an anti-FcRn antibody or fragment thereof.

Advantageous effects

Antibodies of the invention or fragments thereof that are specific for FcRn, which is a receptor with high affinity for IgG, cause little or no immunogenicity-related problems, and bind FcRn in a manner that does not compete with IgG to reduce serum autoantibody levels, have high affinity and specificity. By virtue of such properties, the antibodies or fragments thereof are useful in the treatment and diagnosis of autoimmune diseases.

Drawings

FIG. 1 shows the results of analysis of antibody expression in CHO-S cells and analysis of HL161A, HL161B, HL C and HL161D antibody proteins obtained by protein A purification on SDS-PAGE gels under reducing or non-reducing conditions. The results showed that each HL161 antibody had a full human IgG1 type structure of about 160kDa in size under non-reducing conditions, and about 55kDa in heavy chain size and about 25kDa in light chain size under reducing conditions, indicating that the antibody consisted of conventional antibody subunits. In fig. 1, lane 1 represents a molecular weight (m.w.) marker, lane 2 represents 2 μg of non-reduced (NEM treated) antibody, and lane 3 represents 2 μg of reduced antibody.

Figure 2 shows the results of analysis using the SPR system in order to determine the Kinetic Dissociation (KD) of 4 anti-FcRn antibodies (HL 161A, HL161B, HL161C and HL 161D) that bind FcRn. The results of fig. 2 were obtained by analyzing the interactions between human FcRn and HL161A, HL161B, HL161C or HL161D antibodies at pH 6.0 and pH 7.4 using the Proteon GLC chip and the Proteon XPR36 (Bio-Rad) system:

figure 2a shows the results of an analysis of the interaction between human FcRn and HL161A antibodies at pH 6.0.

Figure 2b shows the results of an analysis of the interaction between human FcRn and HL161A antibodies at pH 7.4.

Figure 2c shows the results of an analysis of the interaction between human FcRn and HL161B antibodies at pH 6.0.

Figure 2d shows the results of an analysis of the interaction between human FcRn and HL161B antibodies at pH 7.4.

Figure 2e shows the results of an analysis of the interaction between human FcRn and HL161C antibodies at pH 6.0.

Figure 2f shows the results of an analysis of the interaction between human FcRn and HL161C antibodies at pH 7.4.

FIG. 2g shows the results of an analysis of the interaction between human FcRn and HL161D antibodies at pH 6.0.

Figure 2h shows the results of an analysis of the interaction between human FcRn and HL161D antibodies at pH 7.4.

Figure 3 shows the ability of two antibodies selected to bind to the cell surface and shows the results obtained by treating HEK293 cells overexpressing human FcRn with HL161A and HL161B antibodies selected to bind to human FcRn present on the surface of HEK293 cells and analyzing the binding of antibodies to the cell surface at pH 6.0 and pH 7.4. Binding of each of the HL161A and HL161B antibodies to human FcRn was expressed as MFI values obtained by Fluorescence Activated Cell Sorting (FACS) using Alexa 488-labeled anti-human goat antibodies after treatment of cells with each antibody at different pH.

Figure 4 shows the results of an analysis of the ability to block human IgG from binding to cells expressing human FcRn at pH 6.0 and shows the observation whether binding of two selected antibodies to cell surface human FcRn can block human IgG from binding to human FcRn at the cellular level. By serial 4-fold dilutions from 200nM of each of HL161A and HL161B antibodies that were confirmed to bind to HEK293 cells overexpressing human FcRn, a curve was obtained that blocked the ability of Alexa 488-labeled human IgG to bind to human FcRn.

FIGS. 5a and 5B show the results of an analysis of the effect of an HL161A antibody and an HL161B antibody on IgG1 catabolism selected from transgenic mice expressing human FcRn Tg32 (hFcRn+/+, hβ2m+/+, mFcRn-/-, mβ2m-/-). At 0 hours, 5mg/kg biotin-hIgG and 495mg/kg human IgG were intraperitoneally administered into the body to saturate IgG. For drug administration, igG1, HL161A, HL161B or PBS were intraperitoneally injected at doses 5, 10 and 20mg/kg once a day 24, 48, 72 and 96 hours after biotin-IgG administration. Sample collection was performed 24, 48, 72, 96, 120 and 168 hours after biotin-IgG administration. At 24, 48, 72 and 96 hours, blood was collected before drug administration, and the remaining amount of biotin-IgG was analyzed by ELISA method. The results are expressed as the ratio of the remaining amount at each time point to the remaining amount of 100% in the blood sample collected at 24 hours.

Fig. 6 shows the results of analysis of monkey IgG blood level changes caused by administration of two antibodies (HL 161A and HL 161B) to cynomolgus monkeys having 96% sequence homology to human FcRn. HL161A antibody and HL161B antibody were each intravenously administered to cynomolgus monkeys at doses of 5 and 20mg/kg once a day, and the results showed up to a 70% reduction in monkey IgG compared to 0 hours, and about a 30% reduction up to day 29.

Figure 6a shows serum IgG reduction effect of HL161A and HL161B antibodies at different antibody concentrations.

FIG. 6B shows serum IgG reduction by HL161A and HL161B antibodies (concentration: 5 mg/kg) in monkey individuals.

FIG. 6c shows serum IgG reduction by HL161A and HL161B antibodies (concentration: 20 mg/kg) in monkey individuals.

FIGS. 7a and 7b show access to a cynomolgus monkey in useResults of pharmacokinetic profiles of HL161A and HL161B were analyzed in the experiments of the row. HL161B overall showed high half-life AUC and C compared to HL161A _max 。

Fig. 8a to 8c show the results of analyzing the change in blood levels of monkey IgM, igA and albumin caused by administration of HL161A antibody and HL161B antibody in experiments performed using cynomolgus monkeys. There were slight changes in the blood levels of monkey IgM, igA and albumin, which were within the normal range of cynomolgus monkeys, indicating that such changes were due to differences between individuals, not the effects of the test substances.

FIG. 8a shows changes in serum IgM levels in monkeys.

FIG. 8b shows changes in serum IgA levels in monkeys.

Fig. 8 shows changes in serum albumin levels in monkeys.

Detailed description of the preferred embodiments

To achieve the above objects, the present disclosure provides an antibody that can specifically bind to FcRn with high affinity in a pH-independent manner and consists of human-derived sequences and thus causes little or no immune response upon in vivo administration.

Antibodies of the present disclosure are binding molecules specific for FcRn. Antibodies can include monoclonal antibodies (e.g., full length antibodies with immunoglobulin Fc domains), antibody compositions with multi-epitope specificity, bispecific antibodies, diabodies, and single chain molecules, as well as antibody fragments (e.g., fab, F (ab') ₂ And Fv), but is not limited thereto. Antibodies according to the present disclosure may for example be monoclonal antibodies against human FcRn.

Monoclonal antibodies include murine antibodies. In addition, monoclonal antibodies include "chimeric antibodies" and fragments of such antibodies in which portions of the heavy and/or light chains are identical or homologous to corresponding sequences in antibodies derived from a particular species (e.g., murine) or belonging to a particular antibody class or subclass, while the remainder of the chains are identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass (e.g., human), so long as they exhibit the desired biological activity. "humanized antibodies" are used as downstream collections of "chimeric antibodies".

As an alternative to humanization, human antibodies may be produced. A "human antibody" is an antibody that is produced by a human or has an amino acid sequence that corresponds to an antibody produced using any of the human antibody production techniques. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but whose endogenous locus has been disabled, e.g., immunized xenomice. The antibodies of the disclosure may be, for example, in the form of human antibodies.

Natural four-chain antibodies are heterotetrameric glycoproteins composed of two identical light chains (L) and two identical heavy chains (H). Each light chain has a variable domain at one end (V _L ) And has a constant domain at the other end. At the N-terminus, each heavy chain has a variable domain (V _H ) And has 3 constant domains for the alpha and gamma chains (C _H ) There are 4 CH domains for the μ and ε isoforms.

The term "variable" refers to the fact that certain parts of the variable domains differ widely in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, variability is concentrated in three segments, called hypervariable regions (HVRs), the CDRs in the light chain variable domain and the heavy chain variable domain. The more highly conserved parts of the variable domains are called Framework Regions (FR). The light chain variable domain and the heavy chain variable domain comprise domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from the N-terminus to the C-terminus.

In the present disclosure, antibodies with affinity and specificity for human FcRn are obtained using human immunoglobulin transgenic animals. Transgenic animals can be produced by inactivating an Ig germline gene of the animal and implanting a human Ig germline gene locus. The use of transgenic animals has the following advantages: antibodies are naturally optimized by the animal immune system without the need for affinity maturation, so that antibody drugs with low immunogenicity and high affinity can be developed in a short time (US 20090098134,US 20100212035,Menoret et al, eur J Immunol,40:2932, 2010).

In the present disclosure omniRat, a technique of the patent of human immunoglobulin transgenic rat, is used ^TM (OMT, USA). OmniRat ^TM Antibodies with high affinity for human FcRn can be efficiently selected because it has heavy chains composed of CH2 and CH3 domains from the rat gene and V, D and J regions and CH1 domains from the human gene, as well as kappa and lambda light chains from humans, thereby efficiently selecting antibodies with high affinity for human FcRn (meroret et al, eur J Immunol,40:2932, 2010).

In order to obtain monoclonal antibodies with high affinity for FcRn, the human FcRn-immunized transgenic rats (omniray ^TM ) And then B cells are extracted from the cells and fused with myeloma cells to produce hybridomas, after which the produced antibodies are purified from the produced hybridomas.

Antibodies of the present disclosure act as non-competitive IgG inhibitors that bind to FcRn. Binding of the antibodies of the present disclosure to FcRn results in inhibition of the pathogenic antibody to FcRn, which facilitates clearance (i.e., removal) of the pathogenic antibody from the body of the subject, thereby reducing the half-life of the pathogenic antibody.

As used herein, the term "pathogenic antibody" means an antibody that causes a pathological condition or disease. Examples of such antibodies include, but are not limited to, anti-platelet antibodies, anti-acetylcholine antibodies, anti-nucleic acid antibodies, anti-phospholipid antibodies, anti-collagen antibodies, anti-ganglioside antibodies, anti-desmosome antibodies, and the like.

An advantage of the antibody or fragment thereof of the present disclosure is that it can non-competitively inhibit binding of a pathogenic antibody to FcRn at physiological pH (i.e., pH 7.0-7.4). FcRn binds to its ligand (i.e., igG) and exhibits substantially no affinity for IgG at physiological pH rather than acidic pH. Thus, an anti-FcRn antibody that specifically binds to FcRn at physiological pH acts as a non-competitive inhibitor of IgG binding to FcRn, and in this case, the binding of the anti-FcRn antibody to FcRn is not affected by the presence of IgG. Thus, antibodies of the invention that bind FcRn in a pH-independent manner non-competitively to IgG have the advantage over conventional competitive inhibitors (i.e., antibodies that bind FcRn competitively to IgG): it can treat diseases even at significantly low concentrations by FcRn-mediated IgG signaling. Furthermore, during intracellular movement in the state of binding to FcRn, the anti-FcRn antibodies of the present disclosure maintain their binding to FcRn with higher affinity than blood IgG, and thus can inhibit IgG binding to FcRn even in endosomes (acidic pH environment where IgG can bind to FcRn), thereby promoting clearance of IgG.

The antibodies of the present disclosure have affinity for FcRn even in physiological pH environments (i.e., pH 7.0-7.4) where IgG does not bind FcRn. At pH 6.0, the antibody of the present disclosure has a higher affinity for FcRn than serum IgG, showing that it acts as a non-competitive inhibitor.

In one embodiment of the disclosure, the disclosure relates to an antibody or fragment thereof that specifically binds to FcRn comprising:

Those of skill in the art will appreciate that deletion, addition, or substitution of some amino acids within the amino acid sequences set forth in the above SEQ ID nos are also within the scope of the present disclosure.

In addition, sequences having homology within a certain range to the nucleotide sequences and amino acid sequences described in the present disclosure are also within the scope of the present disclosure. "homology" refers to similarity to at least one nucleotide or amino acid sequence selected from SEQ ID No. 1 to 44, and includes at least 90% homology. Preferably, the homology may be at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. Homology comparisons are performed visually or at standard settings using known comparison programs such as the BLAST algorithm. Commercially available programs can express the homology between two or more sequences as a percentage. Homology (%) can be calculated for adjacent sequences.

In addition, antibodies that specifically bind to FcRn having KD (dissociation constant) 0.01-2nM at pH 6.0 and pH 7.4 are also within the scope of the present disclosure. "KD" as used herein refers to the equilibrium dissociation constant of antibody-antigen binding and can be calculated using the following equation: kd=kd/ka, where ka represents the binding rate constant and KD represents the dissociation rate constant. Measurement of kd or ka can be performed at 25℃or 37 ℃.

In one example, an antibody of the disclosure comprises: CDR1 comprising the amino acid sequence of SEQ ID No. 21, CDR2 comprising the amino acid sequence of SEQ ID No. 22 and CDR3 comprising the amino acid sequence of SEQ ID No. 23,

CDR1 comprising the amino acid sequence of SEQ ID No. 27, CDR2 comprising the amino acid sequence of SEQ ID No. 28 and CDR3 comprising the amino acid sequence of SEQ ID No. 29,

CDR1 comprising the amino acid sequence of SEQ ID No. 33, CDR2 comprising the amino acid sequence of SEQ ID No. 34 and CDR3 comprising the amino acid sequence of SEQ ID No. 35, or

CDR1 comprising the amino acid sequence of SEQ ID No. 39, CDR2 comprising the amino acid sequence of SEQ ID No. 40 and CDR3 comprising the amino acid sequence of SEQ ID No. 41.

The amino acid sequences described in the above SEQ ID No may be amino acid sequences corresponding to CDR1 through CDR3 of the heavy chain variable region.

In another example, an antibody or antigen binding fragment of the disclosure comprises:

CDR1 comprising the amino acid sequence of SEQ ID No. 24, CDR2 comprising the amino acid sequence of SEQ ID No. 25 and CDR3 comprising the amino acid sequence of SEQ ID No. 26,

CDR1 comprising the amino acid sequence of SEQ ID No. 30, CDR2 comprising the amino acid sequence of SEQ ID No. 31 and CDR3 comprising the amino acid sequence of SEQ ID No. 32,

CDR1 comprising the amino acid sequence of SEQ ID No. 36, CDR2 comprising the amino acid sequence of SEQ ID No. 37 and CDR3 comprising the amino acid sequence of SEQ ID No. 38, or

CDR1 comprising the amino acid sequence of SEQ ID No. 42, CDR2 comprising the amino acid sequence of SEQ ID No. 43 and CDR3 comprising the amino acid sequence of SEQ ID No. 44.

The amino acid sequences set forth in the above SEQ ID No may be amino acid sequences corresponding to CDR1 through CDR3 of the light chain variable region.

In particular, the antibodies or antigen binding fragments of the present disclosure comprise: a heavy chain variable region and a light chain variable region selected from one or more of the following:

a heavy chain variable region comprising CDR1 comprising the amino acid sequence of SEQ ID No. 21, CDR2 comprising the amino acid sequence of SEQ ID No. 22 and CDR3 comprising the amino acid sequence of SEQ ID No. 23, and a light chain variable region comprising CDR1 comprising the amino acid sequence of SEQ ID No. 24, CDR2 comprising the amino acid sequence of SEQ ID No. 25 and CDR3 comprising the amino acid sequence of SEQ ID No. 26;

A heavy chain variable region comprising CDR1 comprising the amino acid sequence of SEQ ID No. 27, CDR2 comprising the amino acid sequence of SEQ ID No. 28 and CDR3 comprising the amino acid sequence of SEQ ID No. 29, and a light chain variable region comprising CDR1 comprising the amino acid sequence of SEQ ID No. 30, CDR2 comprising the amino acid sequence of SEQ ID No. 31 and CDR3 comprising the amino acid sequence of SEQ ID No. 32;

a heavy chain variable region comprising CDR1 comprising the amino acid sequence of SEQ ID No. 33, CDR2 comprising the amino acid sequence of SEQ ID No. 34 and CDR3 comprising the amino acid sequence of SEQ ID No. 35, and a light chain variable region comprising CDR1 comprising the amino acid sequence of SEQ ID No. 36, CDR2 comprising the amino acid sequence of SEQ ID No. 37 and CDR3 comprising the amino acid sequence of SEQ ID No. 38; and

a heavy chain variable region comprising CDR1 comprising the amino acid sequence of SEQ ID No. 39, CDR2 comprising the amino acid sequence of SEQ ID No. 40 and CDR3 comprising the amino acid sequence of SEQ ID No. 41, and a light chain variable region comprising CDR1 comprising the amino acid sequence of SEQ ID No. 42, CDR2 comprising the amino acid sequence of SEQ ID No. 43 and CDR3 comprising the amino acid sequence of SEQ ID No. 44.

In one example, an antibody or antigen binding fragment of the disclosure comprises one or more heavy chain variable regions and a light chain variable region comprising one or more amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID nos. 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.

In particular, the antibodies or antigen binding fragments of the present disclosure comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID nos. 2, 4, 6, 8 or 10, and/or a light chain variable region comprising the amino acid sequence of SEQ ID nos. 12, 14, 16, 18 or 20.

In detail, the antibodies or antigen binding fragments of the present disclosure comprise one or more heavy chain variable regions and light chain variable regions selected from the group consisting of:

a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 2 and a light chain variable region comprising the amino acid sequence of SEQ ID No. 12;

a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 4 and a light chain variable region comprising the amino acid sequence of SEQ ID No. 14;

a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 6 and a light chain variable region comprising the amino acid sequence of SEQ ID No. 16;

a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 8 and a light chain variable region comprising the amino acid sequence of SEQ ID No. 18; and

a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 10 and a light chain variable region comprising the amino acid sequence of SEQ ID No. 20.

As these terms are used herein to refer to antibodies, "fragment" or "antibody fragment" refers to a polypeptide (e.g., an antibody heavy or light chain polypeptide) derived from an antibody polypeptide molecule that does not include a full-length antibody polypeptide, but still comprises at least a portion of a full-length antibody polypeptide. Antibody fragments often comprise polypeptides that comprise a cleavage portion of a full-length antibody polypeptide, although the term is not limited to such cleaved fragments. As fragments are used herein to refer to antibodies that encompass fragments comprising a single polypeptide chain derived from an antibody polypeptide (e.g., a heavy chain or light chain antibody polypeptide), it is understood that the antibody fragment itself may not bind an antigen.

Fragments of antibodies of the present disclosure include, but are not limited to, single chain antibodies, bispecific, trispecific and multispecific antibodies such as diabodies, triabodies and tetrabodies, fab fragments, F (ab') ₂ Fragments, fd, scFv, domain antibodies, bispecific antibodies, miniantibodies, scaps (sterol-modulating binding protein cleavage activating protein), chelating recombinant antibodies, trisomy or tetrasomy, intracellular antibodies, nanobodies, small Modular Immunopharmaceuticals (SMIPs), binding domain immunoglobulin fusion proteins, camelized antibodies, VHH-containing antibodies, igD antibodies, igE antibodies, igM antibodies, igG1 antibodies, igG2 antibodies, igG3 antibodies, igG4 antibodies, derivatives in antibody constant regions, and synthetic antibodies based on protein scaffolds with the ability to bind FcRn. It will be apparent to those skilled in the art that any fragment of an antibody of the present disclosure will exhibit the same properties as an antibody of the present disclosure.

Furthermore, antibodies having mutations in the variable region are included within the scope of the present disclosure. Examples of such antibodies include antibodies having conservative substitutions of amino acid residues in the variable region. As used herein, the term "conservative substitution" refers to a substitution to another amino acid residue that has similar properties as the original amino acid residue. For example, lysine, arginine and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine and methionine have similar properties in that they have nonpolar side chains. In addition, tyrosine, phenylalanine, tryptophan and histidine have similar properties in that they have aromatic side chains. Thus, it will be apparent to those skilled in the art that even when amino acid residues in groups exhibiting similar properties as described above are substituted, it will not exhibit a particular change in properties. Thus, antibodies having mutations in the variable region resulting from conservative substitutions are included within the scope of the present disclosure.

Furthermore, the antibodies or fragments thereof of the present disclosure may be used as conjugates with another substance. Substances that may be used as conjugates with the antibodies or fragments thereof of the present disclosure include therapeutic agents commonly used to treat autoimmune diseases, substances capable of inhibiting FcRn activity, and moieties that physically bind to the antibody to improve its stabilization and/or retention in the circulation (e.g., in serum, lymph or other tissues). For example, an antibody that binds FcRn may be bound to a polymer (e.g., a non-antigenic polymer such as a polyalkylene oxide or polyethylene oxide). The weight of suitable polymers varies widely. Polymers having a number average molecular weight of about 200 to about 35,000 (or about 1,000 to about 15,000 and 2,000 to about 12,500) may be used. For example, antibodies that bind FcRn may be conjugated to water-soluble polymers, e.g., hydrophilic polyethylene polymers, such as polyvinyl alcohol and polyvinylpyrrolidone. A non-limiting list of such polymers includes, but is not limited to, polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycol, polyoxyethylated polyols, copolymers thereof, and block copolymers thereof, provided that the water solubility of the block copolymer is maintained.

In another embodiment, the present disclosure relates to a pharmaceutical composition for treating an autoimmune disease, the pharmaceutical composition comprising an anti-FcRn antibody and one or more pharmaceutically acceptable carriers. In addition, the present disclosure also relates to a method of treating an autoimmune disease, the method comprising administering to a patient in need thereof an effective amount of an antibody that specifically binds to FcRn.

The pharmaceutical compositions may comprise pharmaceutically acceptable carriers, excipients, and the like, as are well known in the art. The pharmaceutically acceptable carrier should be compatible with the active ingredient, such as an antibody or fragment thereof of the present disclosure and may be physiological saline, sterile water, ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture of two or more thereof. In addition, the pharmaceutical compositions of the present disclosure may contain other conventional additives including antioxidants, buffers, and bacteriostats, if desired. In addition, the pharmaceutical compositions of the present disclosure may be formulated in injectable form, such as aqueous solutions, suspensions or emulsions, with the aid of diluents, dispersants, surfactants, binders and lubricants. Furthermore, the pharmaceutical compositions of the present disclosure may be provided by formulation into various forms such as powders, tablets, capsules, liquids, injections, ointments, syrups, and the like, as well as single or multi-dose containers such as sealed ampoules or vials.

The pharmaceutical compositions of the present disclosure may be applied to all autoimmune diseases mediated by IgG and FcRn, and common examples of such autoimmune diseases include, but are not limited to, immune neutropenia, guillain-barre syndrome, epilepsy, autoimmune encephalitis, isaac syndrome, nevi syndrome, pemphigus vulgaris, pemphigus larum, bullous pemphigoid, acquired epidermolysis bullosa, gestational pemphigoid, mucosal pemphigoid, antiphospholipid syndrome, autoimmune anemia, autoimmune Grave disease, goodpasture syndrome, myasthenia gravis, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenic purpura, lupus nephritis, and membranous nephropathy.

In the treatment methods of the present disclosure, the dosage of the antibody may be appropriately determined by considering the severity, condition, age, medical history, etc., of the patient. For example, the antibody may be administered at a dose of 1mg/kg to 2 g/kg. The antibody may be administered once or several times.

The present disclosure also provides a method for alleviating an autoimmune disorder or an alloimmune disorder comprising administering an antibody or fragment of an antibody of the present disclosure to a subject in need of treatment. The present disclosure also provides specific anti-FcRn therapies.

The methods of the invention or the anti-FcRn therapies of the invention for alleviating an autoimmune or alloimmune disorder can be achieved by administering the pharmaceutical compositions of the present disclosure to a subject. The pharmaceutical compositions of the present disclosure may be administered orally or parenterally. The pharmaceutical compositions of the present disclosure may be administered by a variety of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, epidural, intracardiac, transdermal, subcutaneous, intraperitoneal, gastrointestinal, sublingual, and topical routes. The dosage of the compositions of the present disclosure may vary depending on a variety of factors, such as patient weight, age, sex, health condition and diet, the time and method of administration, rate of excretion and severity of the disease, and can be readily determined by one of skill in the art. Generally, 1-200mg/kg and preferably 1-40mg/kg of the composition may be administered to a patient suffering from an autoimmune or alloimmune disorder, and these regimens are preferably designed to reduce serum endogenous IgG concentrations to less than 75% of the pre-treatment value. Intermittent and/or long-term (sustained) administration strategies may be employed depending on the condition of the patient.

In another embodiment, the present disclosure also provides a diagnostic composition comprising an antibody or fragment thereof of the present disclosure and a diagnostic method using the diagnostic composition. In other words, antibodies of the present disclosure or fragments thereof that bind FcRn have diagnostic uses in vitro and in vivo.

In another embodiment, the present disclosure relates to a composition for detecting FcRn, the composition comprising an anti-FcRn antibody or fragment thereof. The present disclosure also provides methods, systems, or devices for detecting FcRn in vivo or in vitro comprising treating an anti-FcRn antibody.

An in vitro assay method, system or device may, for example, comprise (1) contacting a sample with an antibody that binds FcRn; (2) Detecting complexes formed between the FcRn-binding antibody and the sample; and/or (3) contacting a reference sample (e.g., a control sample) with the antibody; and (4) determining the extent of complex formation between the antibody and the sample by comparison to a reference sample. A change in complex formation (e.g., a statistically significant change) in the sample or subject, as compared to a control sample or subject, is indicative of the presence of FcRn in the sample.

An in vivo detection method, system or device may include: (1) administering to the subject an antibody that binds FcRn; and (2) detecting the formation of a complex between the FcRn-binding antibody and the subject. Detection may include determining the location or time at which the complex is formed. Antibodies that bind FcRn may be directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound antibodies. Suitable detectable substances include a variety of enzymes, prosthetic groups, fluorescent substances, luminescent substances and radioactive substances. Complex formation between an antibody that binds FcRn and FcRn can be detected by measuring or visualizing antibodies that bind or do not bind to FcRn. Conventional detection assays may be used, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or histoimmunohistochemistry. In addition to labeling antibodies that bind FcRn, samples may also be analyzed for the presence of FcRn by a competition immunoassay that uses a standard labeled with a detectable substance and unlabeled antibodies that bind FcRn. In one example of such an assay, a biological sample, a labeled standard, and an antibody that binds FcRn are combined and the amount of labeled standard that does not bind FcRn is determined. The amount of FcRn in a biological sample is inversely proportional to the amount of labeled standard that is not bound to FcRn.

For detection purposes, the antibodies or fragments thereof of the present disclosure may be labeled with fluorophores and chromophores. Because antibodies and other proteins absorb light with wavelengths up to about 310nm, fluorescent moieties with substantial absorption at wavelengths above 310nm and preferably above 400nm should be selected. Antibodies or fragments thereof of the present disclosure may be labeled with a variety of suitable fluorescent agents and chromophores. One group of fluorescers are xanthene dyes, which include fluorescein and rhodamine. Another group of fluorescent compounds are naphthylamines. Once labeled with a fluorophore or chromophore, the antibody may be used to detect the presence of FcRn or its localization in the sample, for example, using fluorescence microscopy (e.g., confocal or deconvolution microscopy).

The antibodies or fragments thereof of the present disclosure can be used to detect the presence of FcRn or its localization by various methods such as histological analysis, protein arrays, and FACS (fluorescence activated cell sorting).

In the present disclosure, the presence of FcRn or FcRn expressing tissue in vivo may be demonstrated by an in vivo imaging method. The method comprises (i) administering to a subject (e.g., a patient with an autoimmune disease) an anti-FcRn antibody conjugated to a detectable label; and (ii) exposing the subject to means for detecting the detectable label to reveal FcRn expressing tissue or cells. For example, the subject is imaged by NMR or other tomographic means. Examples of labels that can be used for diagnostic imaging include radiolabels, fluorescent labels, positron emitting isotopes, chemiluminescent agents, and enzymatic markers. Radiolabeled antibodies may also be used in vitro diagnostic assays. The specific activity of an isotopically-labeled antibody depends on the half-life of the radiolabel, the isotopic purity, and how the label is incorporated into the antibody.

The disclosure also provides a kit comprising an antibody or fragment thereof that binds to FcRn and instructions for diagnostic use, e.g., the use of an antibody or fragment thereof that binds FcRn to detect FcRn in vitro (e.g., in a sample, e.g., a biopsy sample or cell from a patient with an autoimmune disease) or in vivo (e.g., by imaging a subject). The kit may also contain at least one additional reagent, such as a label or an additional diagnostic agent. For in vivo use, the antibodies may be formulated as pharmaceutical compositions.

In another embodiment, the disclosure relates to polynucleotide sequences encoding antibodies or fragments thereof of the disclosure.

In one example, a polynucleotide sequence encoding an antibody or fragment thereof of the present disclosure is a sequence having at least 90% homology to one or more sequences selected from the group consisting of SEQ ID nos. 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19, or a sequence having more than 90% homology when compared to the sequences mentioned above.

Specifically, the polynucleotide sequence of an antibody or fragment thereof of the present disclosure is a sequence encoding the heavy chain of an antibody of the present disclosure that is SEQ ID No. 1, 3, 5, 7 or 9, and/or a sequence encoding the light chain of an antibody of the present disclosure that is SEQ ID No. 11, 13, 15, 17 or 19.

In another embodiment, the disclosure relates to recombinant expression vectors comprising the polynucleotides, host cells transfected with the recombinant expression vectors, and methods of making antibodies or fragments thereof that specifically bind to FcRn by using the recombinant expression vectors and host cells.

In one embodiment, the antibodies or fragments thereof of the present disclosure are preferably produced by expression and purification using genetic recombination methods. In particular, by expression in separate host cells or simultaneous expression in a single host cell, variable regions encoding antibodies of the invention that specifically bind to FcRn are produced.

As used herein, the term "recombinant vector" refers to an expression vector capable of expressing a protein of interest in a suitable host cell and means a DNA construct comprising the necessary regulatory elements operably linked to express a nucleic acid insert. As used herein, the term "operably linked" means that the nucleic acid expression control sequence is functionally linked to a nucleic acid sequence encoding a protein of interest, thereby performing a universal function. Efficient ligation to recombinant vectors can be performed using gene recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be readily performed using enzymes well known in the art.

Suitable expression vectors that may be used in the present disclosure may include expression regulatory elements such as promoters, operators, start codons, stop codons, polyadenylation signals and enhancers, and membrane-targeted or secreted signal sequences. The start codon and stop codon are generally considered part of the nucleotide sequence encoding the immunogenic target protein and are necessary for functioning in the individual to whom the genetic construct has been administered and must be in frame with the coding sequence. Promoters may be generally constitutive or inducible. Prokaryotic promoters include, but are not limited to, lac, tac, T3 and T7 promoters. Eukaryotic promoters include, but are not limited to, monkey virus 40 (SV 40) promoters, mouse Mammary Tumor Virus (MMTV) promoters, human Immunodeficiency Virus (HIV) promoters such as the HIV Long Terminal Repeat (LTR) promoter, moloney virus promoters, cytomegalovirus (CMV) promoters, epstein Barr Virus (EBV) promoters, rous Sarcoma Virus (RSV) promoters, and promoters from human genes such as human beta-actin, human hemoglobin, human muscle creatine, and human metallothionein. The expression vector may include a selectable marker that allows selection of host cells containing the vector. Genes encoding products that confer selectable phenotypes such as drug resistance, nutrient requirements, cytotoxic drug resistance, or expression of surface proteins are used as universal selectable markers. Since only cells expressing the selectable marker survive the environment of the selective agent treatment, transformed cells can be selected. In addition, the replicative expression vector may include an origin of replication (a specific nucleic acid sequence that initiates replication). Recombinant expression vectors that may be used in the present disclosure include a variety of vectors such as plasmids, viruses, and cosmids. The kind of the recombinant vector is not particularly limited and the recombinant vector may function in various host cells such as prokaryotic cells and eukaryotic cells to express a gene of interest and produce a desired protein. However, it is preferable to use a vector which can produce a large amount of foreign proteins similar to the natural proteins while having strong expression ability using a promoter exhibiting strong activity.

In the present disclosure, a variety of expression host/vector combinations may be used to express the antibodies or fragments thereof of the present disclosure. For example, expression vectors suitable for eukaryotic hosts include, but are not limited to, SV40, bovine papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus, and retroviruses. Expression vectors that can be used in bacterial hosts include bacterial plasmids such as pET, pRSET, pBluescript, pGEX T, pUC, colE1, pCR1, pBR322, pMB9 and derivatives thereof, plasmids with a broad host range such as RP4, phage DNA represented by a variety of phage lambda derivatives such as gt10, gt11 and NM989, and other DNA phages such as M13 and filamentous single-stranded DNA phages. Expression vectors useful in yeast cells include 2 μm plasmids and derivatives thereof. An expression vector that may be used in insect cells is pVL941.

The recombinant vector is introduced into a host cell to form a transformant. Host cells suitable for use in the present disclosure include prokaryotic cells such as E.coli, bacillus subtilis (Bacillus subtilis), streptomyces sp, pseudomonas sp, proteus mirabilis (Proteus mirabilis) and Staphylococcus sp, fungi such as Aspergillus sp, yeasts such as Pichia pastoris, saccharomyces cerevisiae (Saccharomyces cerevisiae), schizosaccharomyces sp and Neurospora crassa Neurospora crassa, eukaryotic cells such as lower eukaryotic cells and other higher eukaryotic cells such as insect cells.

Host cells that may be used in the present disclosure are preferably derived from plants and mammals, and examples thereof include, but are not limited to, monkey kidney cells (COS 7), NSO cells, SP2/0, chinese Hamster Ovary (CHO) cells, W138, baby Hamster Kidney (BHK) cells, MDCK, myeloma cells, huT78 cells, and HEK293 cells. Preferably CHO cells are used.

In the present disclosure, transfection or transformation into a host cell includes any method by which nucleic acid may be introduced into an organism, cell, tissue or organ, and may be performed using suitable standard techniques selected according to the host cell type, as known in the art. These methods include, but are not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO) ₄ ) Precipitated, calcium chloride (CaCl) ₂ ) Precipitation, agitation with silicon carbide fibers, and agrobacterium-mediated, PEG-mediated, dextran sulfate-mediated, lipofectamine (lipofectamine) -mediated, and dehydration/inhibition-mediated transformations.

The FcRn-specific antibody or fragment thereof of the present disclosure may be produced in large amounts by culturing a transformant comprising the recombinant vector in a nutrient medium, and the medium and culture conditions used in the present disclosure may be appropriately selected according to the kind of host cell. During the culturing process, conditions including temperature, medium pH and culturing time may be controlled so as to be suitable for cell growth and mass production of proteins. Antibodies or antibody fragments produced by recombinant methods as described can be collected from culture media or cell lysates and isolated and purified by conventional biochemical separation techniques (Sambrook et al, molecular Cloning: A laborarory Manual, 2 nd edition, cold Spring Harbor Laboratory Press (1989); deuscher, M., guide to Protein Purification Methods Enzymology, volume 182, academic Press. Inc., san Diego, calif. (1990)). These techniques include, but are not limited to, electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (ion exchange chromatography, affinity chromatography, immunoadsorption chromatography, size exclusion chromatography, etc.), isoelectric focusing, and various modifications and combinations thereof. Preferably, protein a is used to isolate and purify the antibody or antibody fragment.

The antibodies of the present disclosure exhibit an antigen binding capacity of about 300pM or less to about 2nM or less (KD value) at pH 7.4, and also exhibit a KD value of 2nM or less to 900pM or less at pH 6.0. The antibodies of the present disclosure have a strong hFcRn binding affinity of 0.01-2nM and thus it is believed that antibodies that bind to the outside of cells even maintain their binding to endosomes, suggesting that these antibodies have an excellent effect of blocking the binding of autoantibodies to hFcRn. Furthermore, the effect of this blocking of autoantibodies binding to hFcRn was also demonstrated in blocking assays and FACS using cells expressing human FcRn.

Examples

Hereinafter, the present disclosure will be described in further detail with reference to examples. It will be apparent to those of ordinary skill in the art that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present disclosure.

Example 1 construction of an anti-FcRn expressing library Using transgenic rats

Total six transgenic rats were usedOMT) for immunization. Human FcRn was used as immunogen. Two footpads of the rat were immunized 8 times with 0.0075mg human FcRn (each) with adjuvant at 3-day intervals for 24 days. On day 28, rats were immunized with 5-10 μg of immunogen diluted in PBS buffer. On day 28, rat serum was collected and used to measure antibody titers. On day 31, rats were euthanized and popliteal and inguinal lymph nodes were recovered for fusion with P3X63/AG8.653 myeloma cells.

ELISA assays were performed to measure antibody titers in rat serum. Specifically, human FcRn was diluted in PBS (pH 6.0 or pH 7.4) buffer to form a 2 μg/mL solution, and 100 μl of the solution was coated on each well of a 96-well plate, and then incubated at 4 ℃ for at least 18 hours. Each well was washed 3 times with 300 μl wash buffer (PBS containing 0.05% tween-20) to remove unbound human FcRn, and then 200 μl of blocking buffer was added to each well and incubated for 2 hours at room temperature. The test serum samples were diluted 1/100, and then the solutions were serially diluted 2-fold to prepare 10 total test samples having a dilution of 1/100 to 1/256,000. After blocking, each well was washed with 300 μl wash buffer, and then test samples were added to each well and incubated for 2 hours at room temperature. After 3 washes, 100 μl of a 1:50,000 dilution of the second detection antibody in PBS buffer was added to each well and incubated for 2 hours at room temperature. After washing 3 more times, 100 μl of TMB solution was added to each well and allowed to react at room temperature for 10 minutes, and then 50 μl of a stop solution containing 1M sulfuric acid was added to each well to terminate the reaction, after which the OD value at 450nm was measured with a microplate reader. anti-hFcRn IgG titers generated by immunization were higher than those in preimmune serum of non-immunized rats (OD value of 1.0 or higher at 450nm at 1/100 dilution), which showed that the rats were sufficiently immunized.

Using polyethylene glycol, a total of three fused hybridoma libraries A, B and C were generated. Specifically, transgenic rats 1 and 5 were used to generate hybridoma library a, rats 2 and 6 were used to generate hybridoma library B, and rats 3 and 4 were used to generate hybridoma library C. The hybridoma library fusion mixtures used to construct each hybridoma library were cultured in the HAT-containing medium for 7 days, thereby selecting only cells fused to HAT. Hybridoma cells that survived HAT medium were collected and cultured in HT medium for about 6 days, and then supernatant was collected, and the amount of rat IgG in the supernatant was measured using rat IgG ELISA kit (RD-biotech). Specifically, each sample was diluted 1:100, and 100 μl of the dilution was added to each well of the ELISA plate and mixed with peroxidase conjugated anti-rat IgG, followed by reaction at room temperature for 15 minutes. 100. Mu.L of TMB solution was added to each well and allowed to react at room temperature for 10 minutes, and then 50. Mu.L of a stop solution containing 1M sulfuric acid was added to each well to terminate the reaction. Next, the OD value at 450nm was measured with a microplate reader.

Example 2: evaluation of antigen binding affinity and IgG binding action blocking Capacity of anti-hFcRn antibodies of hybridoma library

To analyze the binding of antibodies to human FcRn, the same ELISA assays (pH 6.0 and pH 7.4) as mentioned above were performed. The results of the evaluation of hFcRn binding by the three hybridoma libraries A, B and C showed that hFcRn binding affinities increased in order of a > C > B at both pH6.0 and pH 7.4.

The culture supernatants of the three hybridoma libraries were used to evaluate hFcRn binding affinities at 5ng/mL and 25ng/mL by FACS at pH6.0 and pH 7.4. HEK293 cells stably expressing human FcRn were detached from the flask and subsequently suspended in reaction buffer (0.05% bsa in PBS, pH6.0 or pH 7.4). Diluting the suspension to a cell density of 2X 10 ⁶ Individual cells/mL, and 50 μl of dilution was added to each well of the 96-well plate. Subsequently, 50 μl of hybridoma library culture supernatant, diluted to 10ng/mL and 50ng/mL each, was added to each well and suspended to allow antibody binding. A488 rabbit anti-IgG goat antibody was diluted 1:200 in reaction buffer and 100. Mu.L of the dilution was added to each well and mixed with cell pellet for binding reaction, and then 150. Mu.L of reaction buffer was added to each well. Measurements were performed in FACS (BD). As with ELISA results, it can be seen that hybridoma library A showed the highest binding affinity.

Human FcRn blocking ability of the hybridoma library was assessed by FACS at ph 6.0. Specifically, completely new HEK293 cells and HEK293 cells overexpressing human FcRn were floating in reaction buffer (0.05% bsa in PBS, ph 6.0). Will be 1x 10 ⁵ Individual cells were added to 96-well plates and treated with 10-fold dilutions of 4nM and 0.4nM of each hybridoma library culture supernatant, respectively. To confirm the hIgG blocking capacity, 100nMA 488-hIgG1 was added to each well and then incubated on ice for 90 minutes. After the end of the reaction, the cell pellet was washed with 100 μl of reaction buffer and transferred to a U-round bottom tube, followed by measurement in FACS. Measurement of 100nM A488-The amount of hIgG1, and then blocking (%). The previously formed HL161-1Ag antibody was used as isotype control and as positive control to comparative evaluate the blocking effect of the antibody. Each control was analyzed at a concentration of 1. Mu.M and 2. Mu.M, and hybridoma library samples were measured at two concentrations of 0.4nM and 4 nM. As a result, it was found that hybridoma library A showed the highest blocking effect.

Example 3: isolation of hybridoma clones and selection of human antibodies by FACS

Using hybridoma library a, which showed the highest binding affinity and blocking for human FcRn, each clone was isolated by FACS (flow cytometry), thus obtaining a total of 442 single clones. Isolated monoclonal was cultured in HT medium and the supernatant was collected. Antibody-expressing hybridoma clones that bound to hFcRn in the supernatant were selected by FACS. As a result, it can be seen that 100 clones (M1-M100) strongly bound to hFcRn expressing HEK293 cells.

RNA was isolated from 100 monoclonal selected by FACS analysis and the isolated RNA was sequenced. In one-step sequencing, 88 clones out of 100 were sequenced and these clones were divided into a total of 35 groups (G1 to G38) according to amino acid sequence. Culture supernatants of 33 representative clones (excluding the two clones (G33 and G35) for which medium was not available) were diluted at a concentration of 100ng/mL and evaluated for binding affinity to hFcRn by ELISA.

The hFcRn binding affinity was assessed by FACS at pH 6.0 and 7.4 in the same manner as described above. The order of binding affinities of the clones was similar between pH and the binding strength appeared at various levels.

In addition, 33 clones were evaluated for hFcRn blocking by FACS at pH 6.0. Blocking (%) was calculated based on the measured MFI values. Based on the results of analysis of blocking (%) at concentration 1667pM, each clone was divided into four groups in total: group a:70-100%; group B:30-70%; group C:10-30%; and group D:10% or less.

For kinetic analysis of hybridoma clones by SPR, human FcRn was immobilized and hybridoma cultures were subsequently usedAnalysis was performed as an analyte. Most clones, except for a few clones, showed 10 ⁶ M or higher k _on And 10 ^-3 M or lower k _off Values. Taken together, it was shown that all clones had 10 ^-9 To 10 ^-11 KD value of M.

Of the 5 hybridoma clones, 18 cloned genes without N-glycosylation sites or free cysteines in CDR sequences of group a and group B, which were divided according to the analysis result of hFcRn blocking, were transformed into complete human IgG sequences.

Specifically, the Ig BLAST program of the NCBI web page was used to examine amino acid sequence similarity between VH and VL of the 18 selected antibodies and the human germline antibody set.

To clone 18 human antibody genes, restriction enzyme recognition sites were inserted into both ends of the gene in the following manner. Inserting EcoRI/ApaI into the heavy chain variable domain (VH); inserting EcoRI/XhoI into the light chain lambda variable domain (VL (lambda)); ecoRI/NheI restriction enzyme recognition sites were inserted into the light chain kappa variable domain (VL (kappa)). In the case of a light chain variable domain, the light chain lambda variable (VL (lambda)) gene sequence is linked to a human light chain constant (LC (lambda) region gene during gene cloning, and the light chain kappa variable (VL (kappa)) gene sequence is linked to a human light chain constant (LC (kappa) region gene.

When cloning into the pcho1.0 expression vector to express antibodies in animal cells, the light and heavy chain genes were inserted after cleavage with EcoRV, pacI, avrII and BstZ17I restriction enzymes. To check whether the pcho1.0 expression vector containing 18 selected human antibody genes was identical to the synthetic gene sequence, DNA sequencing was performed.

The complete human IgG was expressed using pcho1.0 expression vector containing all antibody light and heavy chain genes as an animal cell expression system. Human antibodies were obtained by transiently transfecting plasmid DNA of each antibody into CHO-S cells and purifying the antibody secreted into the culture medium by protein a column.

Human IgG was injected into hFcRn expressing Tg32 (hfcrn++, hβ2m++, mFcRn-/-, mβ2m-/-) mice (Jackson laboratory), and then 18 human antibodies transformed into human IgG sequences were administered to the mice with the aim of checking whether the antibodies affected catabolism of human IgG.

Based on the results of in vitro assays for binding affinity (KD) to antigen and assays for human FcRn binding affinity and blocking effects by FACS and analysis of catabolism of human IgG in vivo, the 4 most effective acting human anti-FcRn antibody proteins (HL 161A, HL161B, HL161C and HL 161D) were selected (fig. 1). In addition, by substituting asparagine (N) at heavy chain variable framework position 83 of HL161B antibody with lysine (K), HL161BK antibody without N-glycosylation site was prepared. The nucleotide sequences, amino acid sequences, and CDR sequences of the light chain variable region and the heavy chain variable region of each antibody are shown in tables 1, 2, and 3.

TABLE 1 Polynucleotide sequences of selected human FcRn antibody heavy and light chain variable domains

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TABLE 2 amino acid sequences of selected human FcRn antibody heavy and light chain variable domains

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TABLE 3 CDR sequences of selected human FcRn antibody heavy and light chain variable domains

Example 4: antigen binding affinity of HL161A/HL161B/HL161C/HL161D antibodies by SPR measurement

Binding affinity of HL161A, HL161B, HL C and HL161D antibodies was measured in terms of SPR by immobilizing water soluble hFcRn as a ligand onto a protein GLC chip (Bio-Rad) and measuring affinity. Kinetic analysis was performed using the Proteon XPR36 system. shFcRn was immobilized on GLC chip and antibody samples were allowed to react at concentration 5 and sensorgram (sensorgram) results were obtained. In kinetic analysis, the analysis was repeated 6 times at pH 6.0 and pH 7.4 each using a 1:1 langmuir binding model, and the average KD values were calculated. After the fixing step, the chip was activated under EDAC/NHS conditions of 0.5X, 30. Mu.L/min and 300 seconds. For immobilization, shFcRn was diluted to a concentration of 2. Mu.g/mL and 250. Mu.L in acetate buffer (pH 5.5) and the dilutions were allowed to flow on the chip at a rate of 30. Mu.L/min. When an immobilization level of 200-300RU was reached, the reaction was terminated. Subsequently, the deactivation was performed using ethanolamine at a rate of 30. Mu.L/min for 300 seconds. Samples were prepared by serial 2-fold dilutions of each HL161 antibody from 10nM to 5nM, 2.5nM, 1.25nM, 0.625nM, 0.312nM, etc. Sample dilution was performed at each pH using either 1 XPBST (pH 7.4) or 1 XPBST (pH 6.0). For analysis of the samples, the binding was performed at 50. Mu.L/min for 200 seconds and the dissociation step was performed at 50. Mu.L/min for 600 seconds, after which regeneration was performed at 100. Mu.L/min for 18 seconds using glycine buffer (pH 2.5). Kinetic analysis of each sample was repeated 6 times and then the average antigen binding affinity (KD) was measured. The kinetic parameters of the antibodies resulting from the SPR analysis are shown in table 4 below (fig. 2a to 2 h).

TABLE 4 results of antibody kinetic analysis by human FcRn immobilized SPR

Example 5: binding of HL161A/HL161B antibodies to human FcRn by FACS analysis

Binding to FcRn was analyzed by FACS system at each pH using stable HEK293 cells expressing human FcRn. FcRn binding assays were performed using FACS in pH 6.0 and 7.4 response buffer at pH. Specifically, 100,000 human FcRn expressing stable HEK293 cells were washed with PBS buffer and centrifuged at 4500 rpm for 5 min in a tabletop micro centrifuge to obtain cell pellet. Antibodies were added to 100 μl of PBS at pH 6.0 or pH 7.4/10 mM EDTA. The remaining cell pellet was suspended in reaction buffer and cell counts were performed. 10. Mu.L of the cell suspension was added to the slide and the number of cells in the cell suspension was counted in the TC10 system, after which the cell suspension was diluted with reaction buffer to a cell concentration of 2X 10 ⁶ Individual cells/mL. Each antibody sample was diluted to 500nM. For analysis at pH 6.0, dilutions were diluted to 20nM in 96-well V-bottom plates and 50 μl of dilution was added to each well. For analysis at pH 7.4, 500nM antibody samples were serially diluted 3-fold and analyzed at concentrations ranging from 250nM to 0.11 nM. 50 μl was diluted to 2x 10 ⁶ Cells were added to each well and suspended. The plates were enclosed in a shaker at 4℃and rotated at a rotation angle of 15℃and 10 revolutions per minute for 90 minutes. After completion of the reaction, the plate was removed from the shaker and centrifuged at 2000 rpm for 10 minutes, and the supernatant was removed. Goat antibody against A488 anti-hIgGDilutions were made in reaction buffer at 1:200, and 100 μl of antibody dilution was added to each well and suspended. Next, the plate was again enclosed in a shaker at 4 ℃ and rotated at a rotation angle of 15 ° and 10 revolutions per minute for 90 minutes. After completion of the reaction, the plate was removed from the shaker and centrifuged at 2000 rpm for 10 minutes, and the supernatant was removed. After the washing procedure was performed once more, 100 μl of reaction buffer was added to each well to lyse the cell pellet, and the plate was transferred into a blue tube. Next, 200 μl of reaction buffer was added to each well, and then measurements were performed in FACS. FACS measurements were performed under the following conditions: FS 108 volts, SS 426 volts, FL1 324 volts, FL2 300 volts. Using BD FACSDiva ^TM v6.1.3 software (BD Bioscience) analyzed these cells by FACS. The results are expressed as Mean Fluorescence Intensity (MFI) (fig. 3). HL161A and HL161B antibodies showed MFI values of 10.59 and 8.34 at 10nM concentration and ph6.0, respectively. Antibodies showed EC at pH 7.4 and concentrations of 0.11-250nM, respectively, as analyzed by 4-parameter logistic regression using MFI values ₅₀ (50% effective concentration) values were 2.46nM and 1.20nM.

Example 6: analysis of the blocking effect of the HL161A/HL161B antibodies by FACS

HEK293 cells expressing hFcRn on the cell surface were treated with two antibodies that analyzed their binding affinity for human FcRn on the cell surface and blocking effects of the antibodies were examined based on reduced binding of Alexa-Fluo-488-labeled hIgG 1. The analysis procedure was performed as follows.

2mL of 1 xTE was added to each type of brand-new HEK293 cells and stable HEK293 cells overexpressing human FcRn at 37℃with 5% CO ₂ Incubate for 1 min in incubator. Cells were recovered from the flask and 8mL of reaction buffer (pH 6.0) was added thereto, after which the cells were transferred into a 50mL conical tube. The cell suspension was centrifuged at 2000 rpm for 5 minutes to remove the supernatant, and 1mL of reaction buffer (pH 6.0) was added to each cell pellet. Subsequently, the cell suspension was transferred into a new 1.5mL microcentrifuge tube. Next, the cell suspension was centrifuged at 4000 rpm for 5 minutes, and the supernatant was removed. Subsequently, a reaction buffer (pH 6.0) was added to the stockThe cell pellet was left and the cell number of the cell suspension was counted. Finally, the cell suspension is diluted with reaction buffer to a cell concentration of 2.5x10 ⁶ Individual cells/mL.

Each antibody sample was diluted to 400nM and then diluted at 4-fold serial dilutions in 96-well V-bottom plates. 50. Mu.L of sample diluted to a final concentration of 200nM to 0.01nM was added to each well. Subsequently, 10. Mu.L of Alex488-hIgG1 diluted with 1. Mu.M reaction buffer (pH 6.0) was added to each well. Finally, 40. Mu.L of the cell concentration was diluted to 2.5X10 ⁶ Cells were added to each well and suspended. The plates were enclosed in a shaker at 4℃and rotated at a rotation angle of 15℃and 10 revolutions per minute for 90 minutes. After completion of the reaction, the plate was removed from the shaker and centrifuged at 2000 rpm for 10 minutes to remove the supernatant. mu.L of reaction buffer was added to each well to lyse the cell pellet, and the plate was transferred into a blue tube. Subsequently, 200 μl of reaction buffer was added to each well, and measurements were performed in FACS. FACS measurements were performed under the following conditions: FS 108 volts, SS 426 volts, FL1 324 volts, FL2 300 volts. Using BD FACSDiva ^TM v6.1.3 software (BD Bioscience) analyzed these cells by FACS. The results are expressed as Mean Fluorescence Intensity (MFI). MFI of the test group was treated after deduction of the MFI value (background signal) of the cells measured only. The MFI percentage of the tubes containing competitor was calculated relative to 100% of the control tube (Alexa Fluor 488 only, and no competitor).

When the MFI was lower than that of the tubes containing human IgG1 competitor, the competitor antibodies were determined to have a high competition rate. Based on the HL161A antibody and HL161B antibody blocking (%) measured at pH 6.0 and concentration of 0.01-200nM, 4-parameter logistic regression was performed. As a result, it was confirmed that the HL161A antibody and the HL161B antibody exhibited ICs of 0.92nM and 2.24nM, respectively ₅₀ (50% inhibitory concentration) value (fig. 4).

Example 7: testing the role of HL161A/HL161B in mFcRn-/-hFCRN transgenic 32 (Tg 32) mice

Human IgG was injected into Tg32 (hfcrn++, hβ2m+/+, mFcRn-/-, mβ2m-/-) mice expressing human FcRn (Jackson laboratory), and then HL161A and HL161B were administered to the mice along with human IgG, with the aim of checking whether antibodies affected catabolism of human IgG.

HL161A and HL161B antibodies and human IgG (Greencross, IVglobulinS) were dosed at 5, 10 and 20mg/kg for 4 days and stored, and PBS (phosphate buffered saline) buffer (pH 7.4) was used as vehicle and 20mg/kg IgG1 control. Human FcRn Tg32 mice were acclimatized for about 7 days and were given water and food ad libitum. The temperature (23.+ -. 2 ℃), humidity (55.+ -. 5%) and 12 hours light/12 hours dark cycle were automatically controlled. Each animal group consisted of 4 mice. To use human IgG as a tracer, biotin conjugated hIgG was prepared using a kit (Pierce, cat# 21327). At 0 hours, 5mg/kg biotin-hIgG and 495mg/kg human IgG were intraperitoneally administered into the body to saturate IgG. Each drug was intraperitoneally injected at doses of 5, 10, and 20mg/kg once a day 24, 48, 72, and 96 hours after administration of biotin-IgG. To collect blood, mice were lightly anesthetized with isoflurane (JW Pharmaceutical), and blood was then collected from the retroorbital venous plexus 24, 48, 72, 96, 120 and 168 hours after biotin-IgG administration using heparinized microcytosis capillaries (Fisher). At 24, 48, 72 and 96 hours, the drug was administered after blood collection. Immediately after receiving 0.1mL of whole blood in a microcentrifuge tube, the plasma was separated by centrifugation and stored in a-70 ℃ cryocooler (Thermo) until analysis.

The collected blood was analyzed for the level of biotin-hIgG 1 by ELISA in the following manner. Mu.l of neutravidin (Pierce, 31000) was added to a 96-well plate (Costar, catalog number 2592) to a concentration of 1.0. Mu.g/ml, and then coated at 4℃for 16 hours. Plates were washed 3 times with buffer A (0.05% Tween-20, 10mM PBS, pH 7.4) and then incubated for 2 hours at room temperature in PBS (pH 7.4) buffer containing 1% BSA. Next, the plate was washed 3 times with buffer a, and then a neutravidin plate was prepared with PBS (pH 7.4) buffer containing 0.5% bsa, corresponding to 1 μg/ml. Blood samples were serially diluted 500-1000 fold in buffer B (100mM MES,150mM NaCl,0.5% IgG free BSA,0.05% Tween-20, pH 6.0) and 150. Mu.l of the dilution was added to each well of the plate. The added samples were reacted at room temperature for 1 hour. Next, the plates were washed 3 times with buffer a, and then 200 μl of 1nM HRP conjugated anti-human IgG goat antibody was added to each well and incubated for 2 hours at 37 ℃. Next, the plate was washed 3 times with ice-cold buffer B, and then 100 μl of substrate solution tetramethylbenzidine (RnD, catalogue number: DY 999) was added to each well and allowed to react at room temperature for 15 minutes. Mu.l of a 1.0M sulfuric acid solution (Samchun, catalog number: S2129) was added to each well to terminate the reaction, after which the absorbance at 450nm was measured.

After 24 hours (about T of biotin-IgG in mice) _max The method comprises the steps of carrying out a first treatment on the surface of the Before biotin-IgG catabolism occurred) was set at 100% and the percentage of concentration at other time points relative to 24 hour concentration is shown in fig. 10. The results of the analysis showed half-life of vehicle and 20mg/kg IgG1 control to be 103 hours and 118 hours, respectively. However, the blood IgG half-life of the HL161A antibody at the different doses was 30, 23 and 18 hours, which HL161A antibody showed excellent binding affinity and blocking for human FcRn in vitro assays and the fastest IgG catabolism in human FcRn transgenic Tg32 mice. In addition, HL161B antibodies showed IgG half-lives of 41, 22 and 21 hours. This suggests that pH-independent and Fc-non-competing antibodies against hFcRn have the effect of increasing endogenous antibody catabolism (fig. 5a and 5 b).

Example 8: testing the role of HL161A/HL161B in monkeys

Monkeys IgG, igA, igM and albumin levels administered with HL161A and HL161B antibodies were analyzed using cynomolgus monkeys having 96% homology to human FcRn, and Pharmacokinetic (PK) profiles of the antibodies were analyzed.

1) Analysis of changes in immunoglobulin G expression in monkey blood

First, changes in monkey IgG were analyzed by ELISA measurement. 100. Mu.L of anti-human IgG/Fc antibody (BethyLab, A80-104A) was loaded into each well of a 96-well plate (Costar, catalog number 2592) to a concentration of 4.0. Mu.g/mL, and then coated at 4℃for 16 hours. The plates were washed 3 times with wash buffer (0.05% Tween-20, 10mM PBS, pH 7.4) and then incubated with PBS containing 1% BSA (pH 7.4) for 2 hours at room temperature. Standard monkey IgG was used at a concentration of 3.9-500ng/mL and blood samples were diluted 80,000-fold in PBS (pH 7.4) buffer containing 1% BSA, and the dilutions were plated and incubated for 2 hours at room temperature. Next, the plates were washed 3 times with wash buffer, and then 100 μl of a 20,000-fold dilution of anti-hIgG antibody (Biorad, 201005) was loaded into the plates and allowed to react at room temperature for 1 hour. After washing each plate, 100. Mu.L of substrate solution 3,3', 5' -tetramethylbenzidine (RnD, catalog number: DY 999) was charged into the plate and allowed to react at room temperature for 7 minutes, after which 50. Mu.L of 1.0M sulfuric acid solution (Samchun, catalog number S2129) was added to each well to terminate the reaction. For analysis, absorbance (OD) was measured using absorbance readers (MD, model: versamax) at 450nm and 540 nm. The results showed that when HL161A and HL161B antibodies were administered intravenously into cynomolgus monkeys at doses of 5 and 20mg/kg, respectively, the monkey IgG levels decreased in a dose-dependent manner, and HL161 antibodies effectively blocked IgG-FcRn interactions. 5mg/kg HL161A reduced monkey IgG levels to 47.1% at day 9 and 20mg/kg HL161A reduced monkey IgG levels to 29.6% at day 10. 5mg/kg HL161B reduced monkey IgG levels to 53.6% on day 10 and 20mg/kg HL161B reduced monkey IgG levels to 31% on day 9, suggesting that both antibodies showed similar results (table 5 and fig. 6a and 6 c). Furthermore, monkey IgG levels change from intravenous administration of HL161A and HL161B were compared between individuals, and the results showed that monkey IgG levels between individuals decreased in a very similar manner.

TABLE 5 variation in monkey IgG levels (%)

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2) Analysis of pharmacokinetic profile of HL161A/HL161B in monkey blood

Time-dependent pharmacokinetic Profiles (PK) of HL161A and HL161B after intravenous administration were analyzed by competitive ELISA. Specifically, a 2. Mu.g/mL solution of neutravidin was prepared, and 100. Mu.L of this solution was coated on each well of a 96-well plate, and then incubated at 4℃for 18 hours. The plates were washed 3 times with 300 μl of wash buffer (10 mm PBS containing 0.05% tween-20, pH 7.4) and each well was then incubated with PBS (pH 7.4) containing 1% bsa for 2 hours at 25 ℃. Biotinylated hFcRn was diluted to 1 μg/mL with PBS, and then 100 μl of the dilution was added to each well of a 96-well plate and incubated for 1 hour at 25 ℃. Next, the plates were washed 3 times with 300 μl wash buffer to remove unbound hFcRn, and then standard samples (0.156-20 ng/mL) were added to each well and incubated for 2 hours at 25 ℃. Next, the plates were washed 3 times with wash buffer, and then 100 μl of detection antibody at 1:10,000 dilution in PBS was added to each well and incubated for 1.5 hours at 25 ℃. Finally, the plate was washed 3 times, and 100 μl of TMB solution was added to each buffer and incubated at room temperature for 5 minutes, after which 50 μl of 1M sulfuric acid was added as a reaction termination solution to each well to terminate the reaction. Next, absorbance at 450nm was measured with a microplate reader. The analytical results of HL161A and HL161B are shown in table 6 below, and as can be seen therein, the pharmacokinetic profile of the antibody increases in a dose-dependent manner. The half-life (T1/2) of an antibody is about 6-12 days, which is shorter than commonly known antibodies. In addition, the half-life (overall observation), AUC and C of HL161B were confirmed _max Is higher than HL161A (fig. 7a and 7 b).

TABLE 6 analysis of the pharmacokinetic profiles of HL161A and HL161B at different doses

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3) Analysis of IgM and IgA antibody level changes in monkey blood

ELISA assays for measuring IgM and IgA levels in monkey blood were performed in a manner similar to ELISA methods for measuring IgG levels. Specifically, 100 μl of anti-monkey IgM antibody (Alpha Diagnostic, 70033) or IgA antibody (Alpha Diagnostic, 70043) was added to each well of the 96-well plate to a concentration of 2.0 μg/mL, and then coated at 4 ℃ for 16 hours. The plates were washed 3 times with wash buffer (10 mM PBS containing 0.05% Tween-20, pH 7.4) and then incubated with PBS containing 1% BSA (pH 7.4) for 2 hours at room temperature. Standard monkey IgM was analyzed at concentrations of 7.8-1,000ng/mL and IgA was analyzed at concentrations of 15.6-2,000 ng/mL. Blood samples were diluted 10,000-fold or 20,000-fold in PBS (pH 7.4) buffer containing 1% bsa, and dilutions were added to each well and incubated for 2 hours at room temperature. Next, the plate was washed 3 times with a washing buffer, and then 100 μl of each 5000-fold dilution of the anti-monkey IgM secondary antibody (Alpha Diagnostic, 70031) and the anti-monkey IgA secondary antibody (KPL, 074-11-011) was added to each well and allowed to react at room temperature for 1 hour. The plate was finally washed 3 times, and then 100. Mu.L of substrate solution 3,3', 5' -tetramethylbenzidine (RnD, catalogue number: DY 999) was added to each well and allowed to react at room temperature for 7 minutes. Next, 50. Mu.L of 1.0M sulfuric acid solution (Samchun, catalog number: S2129) was added to each well to terminate the reaction. The absorbance of each well was measured with a 450nm and 540nm absorbance reader (MD, model: versamax).

4) Analysis of changes in albumin levels in monkey blood

Analysis of changes in albumin levels in monkey blood was performed using a commercial ELISA kit (Assaypro, catalog number: EKA 2201-1). Briefly, monkey serum was diluted 4000-fold as a test sample, and 25 μl of the dilution was added to each well of a 96-well plate coated with an antibody capable of binding to monkey albumin. mu.L of biotinylated monkey albumin solution was added to each well and incubated for 2 hours at 25 ℃. The plates were washed 3 times with 200 μl wash buffer and then 50 μl 1:100 dilution of streptavidin-peroxidase conjugated antibody was added to each well and incubated for 30 minutes at 25 ℃. The plate was finally washed 3 times and then 50 μl of substrate was added to each well and incubated for 10 minutes at room temperature. Next, 50 μl of the reaction termination solution was added to each well, and absorbance at 450nm was measured. As a result, no clear change in monkey IgM, igA, and albumin levels due to administration of HL161A and HL161B antibodies was observed throughout the test period (fig. 8 a-8 c). Thus, it was concluded that HL161 antibody was only involved in IgG levels and did not affect IgM and IgA levels, suggesting that it would not significantly affect immune decline due to reduced immunoglobulin levels. Furthermore, no significant change in monkey albumin levels was observed throughout the test period, suggesting that HL161A and HL161B antibodies only specifically block IgG-FcRn interactions.

5) Analysis of blood biochemical levels and urine components

Finally, using the sample at test day 14, antibodies were administered for blood biochemical analysis and urine analysis. In addition, blood biochemical markers including aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), alkaline phosphatase (CPK), creatine phosphokinase (creatine phosphokinase), total Bilirubin (TBIL), glucose (GLU), total Cholesterol (TCHO), triglycerides (TG), total Protein (TP), albumin (Alb), albumin/globulin (a/G), blood urea nitrogen (blood urea nitrogen), creatinine (CRE), inorganic Phosphorus (IP), calcium (Ca), sodium (Na), potassium (K), and chloride (Cl) were analyzed using the Hitachi 7180 system. In addition, urine analysis markers including Leucocytes (LEU), nitrate (NIT), urinary cholesterols (URO), protein (PRO), pH, occult Blood (BLO), specific Gravity (SG), ketone bodies (KET), bilirubin (BIL), glucose (GLU) and ascorbic Acid (ASC) were analyzed using the mix U120 system. Although there was a slight level change, the measured levels were within the normal level range for cynomolgus monkeys.

Although the present disclosure has been described in detail with reference to specific features, it will be apparent to one skilled in the art that such description is for the purpose of illustration only and not limitation of the scope of the disclosure. Accordingly, the substantial scope of the present disclosure will be defined by the appended claims and equivalents thereof.

Sequence listing

<110> Han Nuo biopharmaceutical Co., ltd

<120> antibodies that bind FCRN for the treatment of autoimmune diseases

<130> PF-B1783

<140> PCT/KR2015/004424

<141> 2015-04-30

<150> 61/986,742

<151> 2014-04-30

<160> 44

<170> patent In version 3.2

<210> 1

<211> 363

<212> DNA

<213> Artificial sequence (Artifical)

<220>

<223> Hv of HL161A

<400> 1

gaagtgcagc tgctggaatc cggcggaggc ctggtgcagc ctggcggctc tctgagactg 60

tcctgcgccg cctccgagtt caccttcggc agctgcgtga tgacctgggt ccgacaggct 120

cccggcaagg gcctggaatg ggtgtccgtg atctccggct ccggcggctc cacctactac 180

gccgactctg tgaagggccg gttcaccatc tcccgggaca actccaagaa caccctgtac 240

ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgcgc caagaccccc 300

tggtggctgc ggtccccctt cttcgattac tggggccagg gcaccctggt gacagtgtcc 360

tcc 363

<210> 2

<211> 121

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Hv of HL161A

<400> 2

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Phe Thr Phe Gly Ser Cys

20 25 30

Val Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Thr Pro Trp Trp Leu Arg Ser Pro Phe Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 3

<211> 363

<212> DNA

<213> Artificial sequence (Artifical)

<220>

<223> Hv of HL161B

<400> 3

caactgttgc tccaggaatc cggtcctggt cttgtaaagc catctgagac tctctccctt 60

acctgtaccg ttagcggagg aagtctttcc tcaagcttct cctactgggt gtggatcaga 120

cagcctcccg gaaaagggtt ggagtggatt ggcacaatat actactccgg caacacttac 180

tataacccca gcctgaagag caggctgact atctctgtcg acaccagtaa aaatcacttt 240

tctctgaatc tgtcttcagt gaccgcagcc gacaccgccg tgtattattg cgctcggcgc 300

gccgggattc tgacaggcta tctggattca tggggccagg ggacattggt tacagtgtct 360

agt 363

<210> 4

<211> 121

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Hv of HL161B

<400> 4

Gln Leu Leu Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Ser Ser Ser

20 25 30

Phe Ser Tyr Trp Val Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Thr Ile Tyr Tyr Ser Gly Asn Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Asn His Phe

65 70 75 80

Ser Leu Asn Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Arg Ala Gly Ile Leu Thr Gly Tyr Leu Asp Ser Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 5

<211> 363

<212> DNA

<213> Artificial sequence (Artifical)

<220>

<223> HvK of HL161B

<400> 5

cagctgctgc tgcaagaatc cggccctggc ctggtgaaac cctccgagac actgtccctg 60

acctgcaccg tgtccggcgg ctccctgtcc tccagcttct cctactgggt ctggatccgg 120

cagccccctg gcaagggcct ggaatggatc ggcaccatct actactccgg caacacctac 180

tacaacccca gcctgaagtc ccggctgacc atctccgtgg acacctccaa gaaccacttc 240

agcctgaagc tgtcctccgt gaccgccgct gacaccgccg tgtactactg tgccagaagg 300

gccggcatcc tgaccggcta cctggactct tggggccagg gcaccctggt gacagtgtcc 360

tcc 363

<210> 6

<211> 121

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> HvK of HL161B

<400> 6

Gln Leu Leu Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Ser Ser Ser

20 25 30

Phe Ser Tyr Trp Val Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Thr Ile Tyr Tyr Ser Gly Asn Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Asn His Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Arg Ala Gly Ile Leu Thr Gly Tyr Leu Asp Ser Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 7

<211> 354

<212> DNA

<213> Artificial sequence (Artifical)

<220>

<223> Hv of HL161C

<400> 7

caggtgcagc tcgtgcagtc cggcgcagag gtcaaaaagc ctggtgcatc tgtgaaagtg 60

agttgcaagg ctagcggcta cacctttacc ggatgttata tgcattgggt acgccaagcc 120

cccggacaag gcttggaatg gatggggcgt atcaacccaa actctggcgg gactaattac 180

gcccagaagt ttcagggaag ggtgactatg acaagggaca catccatatc caccgcttat 240

atggacctgt ctcgactgcg gtctgatgat acagccgttt attactgcgc cagagactac 300

agcggatgga gcttcgatta ttgggggcag ggtactttgg tcacagtttc aagt 354

<210> 8

<211> 118

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Hv of HL161C

<400> 8

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Cys

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Asp Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Ser Gly Trp Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 9

<211> 369

<212> DNA

<213> Artificial sequence (Artifical)

<220>

<223> Hv of HL161D

<400> 9

cagctgcagt tgcaggagtc aggccccggt ttggttaagc cttctgaaac cctttctctc 60

acatgcacag tatccggtgg ctccatctcc agttcaagtt actactgggg atggatccgg 120

caacccccag gaaaagggct ggagtggatt ggcaatatat attactctgg gtccacctat 180

tacaaccctt ccctgatgag tagagtgacc atcagcgtgg acacaagcaa aaaccaattc 240

agcctgaagc tttctagcgt gaccgctgcc gacacagctg tctattactg tgcccgccag 300

cttagttata actggaatga taggctgttt gattactggg gccaggggac tctcgttaca 360

gtcagcagc 369

<210> 10

<211> 123

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Hv of HL161D

<400> 10

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser

20 25 30

Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Asn Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Met Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Gln Leu Ser Tyr Asn Trp Asn Asp Arg Leu Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 11

<211> 357

<212> DNA

<213> Artificial sequence (Artifical)

<220>

<223> Lv of HL161A

<400> 11

tcttacgtgc tgacccagcc cccctccgtg tctgtggctc ctggccagac cgccagaatc 60

acctgtggcg gcaacaacat cggctccacc tccgtgcact ggtatcagca gaagcccggc 120

caggcccccg tgctggtggt gcacgacgac tccgaccggc cttctggcat ccctgagcgg 180

ttctccggct ccaactccgg caacaccgcc accctgacca tctccagagt ggaagccggc 240

gacgaggccg actactactg ccaagtgcga gactcctcct ccgaccacgt gatcttcggc 300

ggaggcacca agctgaccgt gctgggccag cctaaggccg ctccctccgt gaccctg 357

<210> 12

<211> 119

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Lv of HL161A

<400> 12

Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Thr Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val His

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Arg Asp Ser Ser Ser Asp His

85 90 95

Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys

100 105 110

Ala Ala Pro Ser Val Thr Leu

115

<210> 13

<211> 357

<212> DNA

<213> Artificial sequence (Artifical)

<220>

<223> Lv of HL161B

<400> 13

tcttacgtgc tgacccagtc cccctccgtg tccgtggctc ctggccagac cgccagaatc 60

acctgtggcg gcaacaacat cggctccaag tccgtgcact ggtatcagca gaagcccggc 120

caggcccccg tgctggtggt gtacgacgac tccgaccggc cctctggcat ccctgagcgg 180

ttctccgcct ccaactccgg caacaccgcc accctgacca tctccagagt ggaagccggc 240

gacgaggccg actactactg ccaagtgtgg gactcctcct ccgaccacgt ggtgttcggc 300

ggaggcacca agctgaccgt gctgggccag cctaaggccg ctccctccgt gaccctg 357

<210> 14

<211> 119

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Lv of HL161B

<400> 14

Ser Tyr Val Leu Thr Gln Ser Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Ala Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys

100 105 110

Ala Ala Pro Ser Val Thr Leu

115

<210> 15

<211> 357

<212> DNA

<213> Artificial sequence (Artifical)

<220>

<223> LvK of HL161B

<400> 15

tcttacgtgc tgacccagtc cccctccgtg tccgtggctc ctggccagac cgccagaatc 60

acctgtggcg gcaacaacat cggctccaag tccgtgcact ggtatcagca gaagcccggc 120

caggcccccg tgctggtggt gtacgacgac tccgaccggc cctctggcat ccctgagcgg 180

ttctccgcct ccaactccgg caacaccgcc accctgacca tctccagagt ggaagccggc 240

gacgaggccg actactactg ccaagtgtgg gactcctcct ccgaccacgt ggtgttcggc 300

ggaggcacca agctgaccgt gctgggccag cctaaggccg ctccctccgt gaccctg 357

<210> 16

<211> 119

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> LvK of HL161B

<400> 16

Ser Tyr Val Leu Thr Gln Ser Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Ala Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys

100 105 110

Ala Ala Pro Ser Val Thr Leu

115

<210> 17

<211> 351

<212> DNA

<213> Artificial sequence (Artifical)

<220>

<223> Lv of HL161C

<400> 17

gacatccaga tgacccagtc accatcatcc ctttccgcat ctgtcggaga tagagtgact 60

atcacctgca gggcttctca aggtatttcc aactacctcg cctggttcca gcaaaagcca 120

ggtaaagccc caaagagctt gatctacgcc gcttctagtc tgcagagtgg agttcctagt 180

aagttctccg gctctggcag tggcacagat tttaccttga ccatttccag cctgcagtct 240

gaggatttcg ctacctacta ttgtcagcag tatgacagct atccccccac atttgggggg 300

ggcactaagg tggagataaa acggacagtg gctgcccctt ctgtctttat t 351

<210> 18

<211> 117

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Lv of HL161C

<400> 18

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Lys Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ser Tyr Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile

115

<210> 19

<211> 351

<212> DNA

<213> Artificial sequence (Artifical)

<220>

<223> Lv of HL161D

<400> 19

agctatgagc tgacccagcc tctgagcgta tctgtcgctc tcggccagac agccagaatt 60

acctgtggcg gcaataacat aggatccaaa aatgttcact ggtatcagca aaaacctggc 120

caagctcccg tgctcgtgat ctaccgggac tctaaccgac ccagtggaat ccccgaacgc 180

tttagcggtt ccaactctgg aaatacagct actctgacta tctccagggc tcaggccggg 240

gatgaggccg attactactg ccaggtgtgg gactcaagca cagtggtctt cggcggaggt 300

accaagttga ctgttcttgg gcagccaaag gccgcacctt cagtgaccct g 351

<210> 20

<211> 117

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Lv of HL161D

<400> 20

Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Asn Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Arg Asp Ser Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Thr Val Val

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala

100 105 110

Pro Ser Val Thr Leu

115

<210> 21

<211> 5

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Hv CDR1 of HL161A

<400> 21

Ser Cys Val Met Thr

1 5

<210> 22

<211> 17

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Hv CDR2 of HL161A

<400> 22

Val Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 23

<211> 12

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Hv CDR3 of HL161A

<400> 23

Thr Pro Trp Trp Leu Arg Ser Pro Phe Phe Asp Tyr

1 5 10

<210> 24

<211> 11

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Lv CDR1 of HL161A

<400> 24

Gly Gly Asn Asn Ile Gly Ser Thr Ser Val His

1 5 10

<210> 25

<211> 7

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Lv CDR2 of HL161A

<400> 25

Asp Asp Ser Asp Arg Pro Ser

1 5

<210> 26

<211> 10

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Lv CDR3 of HL161A

<400> 26

Val Arg Asp Ser Ser Ser Asp His Val Ile

1 5 10

<210> 27

<211> 5

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Hv CDR1 of HL161B or HL161BK

<400> 27

Phe Ser Tyr Trp Val

1 5

<210> 28

<211> 16

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Hv CDR2 of HL161B or HL161BK

<400> 28

Thr Ile Tyr Tyr Ser Gly Asn Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 29

<211> 11

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Hv CDR3 of HL161B or HL161BK

<400> 29

Arg Ala Gly Ile Leu Thr Gly Tyr Leu Asp Ser

1 5 10

<210> 30

<211> 11

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Lv CDR1 of HL161B or HL161BK

<400> 30

Gly Gly Asn Asn Ile Gly Ser Lys Ser Val His

1 5 10

<210> 31

<211> 7

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Lv CDR2 of HL161B or HL161BK

<400> 31

Asp Asp Ser Asp Arg Pro Ser

1 5

<210> 32

<211> 11

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Lv CDR3 of HL161B or HL161BK

<400> 32

Gln Val Trp Asp Ser Ser Ser Asp His Val Val

1 5 10

<210> 33

<211> 5

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Hv CDR1 of HL161C

<400> 33

Gly Cys Tyr Met His

1 5

<210> 34

<211> 17

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Hv CDR2 of HL161C

<400> 34

Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 35

<211> 9

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Hv CDR3 of HL161C

<400> 35

Asp Tyr Ser Gly Trp Ser Phe Asp Tyr

1 5

<210> 36

<211> 11

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Lv CDR1 of HL161C

<400> 36

Arg Ala Ser Gln Gly Ile Ser Asn Tyr Leu Ala

1 5 10

<210> 37

<211> 7

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Lv CDR2 of HL161C

<400> 37

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 38

<211> 10

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Lv CDR3 of HL161C

<400> 38

Gln Gln Tyr Asp Ser Tyr Pro Pro Thr Phe

1 5 10

<210> 39

<211> 5

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Hv CDR1 of HL161D

<400> 39

Ser Tyr Tyr Trp Gly

1 5

<210> 40

<211> 16

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Hv CDR2 of HL161D

<400> 40

Asn Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Met Ser

1 5 10 15

<210> 41

<211> 13

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Hv CDR3 of HL161D

<400> 41

Gln Leu Ser Tyr Asn Trp Asn Asp Arg Leu Phe Asp Tyr

1 5 10

<210> 42

<211> 11

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Lv CDR1 of HL161D

<400> 42

Gly Gly Asn Asn Ile Gly Ser Lys Asn Val His

1 5 10

<210> 43

<211> 7

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Lv CDR2 of HL161D

<400> 43

Arg Asp Ser Asn Arg Pro Ser

1 5

<210> 44

<211> 9

<212> PRT

<213> Artificial sequence (Artifical)

<220>

<223> Lv CDR3 of HL161D

<400> 44

Gln Val Trp Asp Ser Ser Thr Val Val

1 5

Claims

1. An isolated anti-FcRn antibody or antigen binding fragment thereof comprising:

a heavy chain variable region comprising a CDR1 having an amino acid sequence as shown in SEQ ID No. 21, a CDR2 having an amino acid sequence as shown in SEQ ID No. 22 and a CDR3 having an amino acid sequence as shown in SEQ ID No. 23, and a light chain variable region comprising a CDR1 having an amino acid sequence as shown in SEQ ID No. 24, a CDR2 having an amino acid sequence as shown in SEQ ID No. 25 and a CDR3 having an amino acid sequence as shown in SEQ ID No. 26.

2. An isolated anti-FcRn antibody or antigen binding fragment thereof comprising:

A heavy chain variable region with an amino acid sequence shown as SEQ ID No. 2 and a light chain variable region with an amino acid sequence shown as SEQ ID No. 12.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody is a monoclonal antibody or a chimeric antibody.

4. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody is a murine antibody or a humanized antibody.

5. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody is a human antibody.

6. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a full-length antibody, fab, F (ab') ₂ Fd, fv, scFv, domain antibodies, bispecific antibodies, minibodies, scaps, multispecific antibodies, intracellular antibodies, small Modular Immunopharmaceuticals (SMIPs) or binding domain immunoglobulin fusion proteins.

7. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody comprises an IgD antibody, igE antibody, igM antibody, igG1 antibody, igG2 antibody, igG3 antibody, igG4 antibody.

8. A polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 7.

9. A polynucleotide encoding an anti-FcRn antibody or antigen binding fragment thereof, comprising:

the sequence encoding the heavy chain variable region of SEQ ID No. 1 and the sequence encoding the light chain variable region of SEQ ID No. 11.

10. A recombinant expression vector comprising the polynucleotide of claim 8 or 9.

11. A host cell transfected with the recombinant expression vector of claim 10.

12. A method of making an anti-FcRn antibody or antigen-binding fragment thereof, comprising:

culturing the host cell of claim 11 and producing antibodies therefrom; and

the antibodies produced are isolated and purified to recover antibodies that specifically bind to FcRn.

13. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 7 and one or more pharmaceutically acceptable carriers.

14. Use of the pharmaceutical composition of claim 13 in the manufacture of a medicament for treating a patient suffering from an autoimmune disease.

15. The use of claim 14, wherein the autoimmune disease is one selected from the group consisting of: immune neutropenia, guillain-Barre syndrome, epilepsy, autoimmune encephalitis, isaac syndrome, nevi syndrome, pemphigus vulgaris, deciduous pemphigus, bullous pemphigoid, acquired epidermolysis bullosa, gestational pemphigoid, mucosal pemphigoid, antiphospholipid syndrome, autoimmune anaemia, autoimmune Grave's disease, goodpasture's syndrome, myasthenia gravis, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenic purpura, lupus nephritis, and membranous nephropathy.

16. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 7 labeled with a detection label.

17. A method of detecting FcRn in vitro for non-diagnostic purposes, the method comprising using the antibody or antigen binding fragment thereof of any one of claims 1 to 7.